The structural origins of specificity in protein-nucleic acid recognition will be investigated using EcoRV endonuclease as the experimental system. A site-directed mutagenesis strategy, based on the known 2.0 A co-crystal structure, will be employed to test current hypotheses regarding the sources of sequence discrimination. Specific questions to be addressed include (i) the role of buried interface water molecules in providing strength and specificity of binding, (ii) the role of distal structural elements in facilitating a required and sequence- specific enzyme conformational change, (iii) the magnitude of the energetic cost associated with a sequence-dependent 50 degree bend in the cognate DNA duplex, which results in a requirement for "indirect readout" of sequence information. The mutational analysis will include thorough characterization of enzyme variants by both kinetic and crystallographic approaches. Crystal structures of mutants will be determined in both unliganded and DNA-complexed states, leading eventually to a database of modified protein-DNA crystal structures of value in corroborating theoretical studies in the field.